PROJECT SUMMARY Sponges are one of the richest sources of bioactive marine natural products with significant therapeutic value. Often the compounds are present in low concentrations which hampers clinical and drug development studies. Increasing evidence suggests that microbes such as bacteria may be the true producers of many of the compounds. There are two biological approaches that can solve this supply problem: 1) identification and heterologous expression of the biosynthetic gene cluster; or 2) identification and culture of the microbial producer. Lasonolide A, isolated from the marine sponge Forcepia sp., exhibits potent and selective cytotoxicity against mesenchymal cancer cells including leukemia, melanomas and glioblastomas. Lasonolide A exerts its function at ultra-low nanomolar level via a unique mode of action by inducing a rapid reversible premature chromosome condensation (PCC) via the activation of RAF1 kinase as demonstrated by Dr. Pommier of NCI, suggesting the compound a good candidate as a new anticancer drug. The Natural Products Branch of NCI is expressing an interest in conducting pre-clinical and clinical evaluations on the compound. The lack of supply of lasonolide A has been a major issue and hampering such evaluation studies. This proposal aims to identify the biosynthetic pathway of lasonolide A, providing the prerequisites for the development of a cost-friendly and sustainable method to supply the compound which will substantially facilitate its evaluation studies as a new anticancer drug. In proposed studies, we will apply both culture-independent, metagenomics, and culture-depend, cultivation, approaches to find genes involved in the biosynthesis of lasonolide A. Specific Aim 1 will use metagenomics to identify potential lasonolide A-biosynthetic genes by analyzing a library prepared from an enriched cell faction rich of lasonolide A or from total sponge Forcepia. Specific aim 2 will screen a total of 1,000 microbial strains in a Forcepia-microbe collection at HBOI to determine whether any strain is able to produce lasonolide A. Identification of such cultivated producer will significantly facilitate the identification of biosynthetic gene cluster, development of a fermentation- based supply of lasonolide A, and further strain improvement studies. This project is expected to develop a set of tools which will be generally useful in approaching biosynthesis of and accessing sustainable supplies for a number of complex supply-limited marine natural products.